The goal of the research program described in this application is the development of a new paradigm in therapeutic chemical design for small molecules to target Hypertrophic Cardiomyopathy (HCM.) Though the disease afflicts roughly 1 in 500 people, and can at times have devastating health consequences, current treatment options remain limited and are largely palliative. With the expansion of genetic testing in the past decade, patients are now identified at earlier stages of the disorder (genotype +, phenotype - ), raising the motivation for directly targeting the primary biophysical disease mechanism prior to the onset of overt disease in order to change the natural history of the disorder. We have developed a robust integrative program aimed at understanding disease mechanism across multiple levels of resolution, from computational models to in vitro systems, to eventual in vivo mouse models of disease. We here propose to identify small molecule modulators targeted at initial molecular insult caused by mutation. Our goal is not the generation of a specific medical treatment, but the development, rather, of a novel, high-throughput approach through this ambitious proof of concept research program. We have identified several general categories of thin filament mutations that are closely linked to both the structure and dynamics of myofilament activation, and how the components interact with each other to create the allosterically regulated cardiac thin filament. Both the overall design of the program is highly innovative in the creation of a new therapeutic development paradigm based on physics, chemistry, and biology, and the integrated methods themselves. The program will be implemented via the following 3 Specific Aims: Aim 1: To utilize our all-atom, explicitly solvated cardiac thin filament model as a platform to identify small molecule compounds that bind the thin filament and increase the flexibility of the TNT1 C-terminal linker domain and to fully validate the effects of this modulation on a known cTnT HCM mutation in vitro and in vivo. Aim 2: To screen for, design and test novel small molecule modulators that target the dynamics of the cTnI N- terminus in silico and validate and test their effects on the kinetics of Ca2+ dissociation from cTnC in vitro and in vivo. Aim 3: To leverage our recently refined, explicitly solvated structure of the cardiac tropomyosin overlap domain of the thin filament to screen for novel modulators of overlap dynamics, specifically targeting hydrogen bonding potential or nonlocal effects as a mediator of flexibility and validating these effects in vitro and in vivo.